DNA Encoding a Plant Lipase, Transgenic Plants and a Method for Controlling Senescence in Plants

ABSTRACT

Regulation of expression of senescence in plants is achieved by integration of a gene or gene fragment encoding senescence-induced lipase into the plant genome in antisense orientation. The carnation and Arabidopsis genes encoding senescence-induced lipase are identified and the nucleotide sequences are used to modify senescence in transgenic plants.

This application is a continuation-in-part of application Ser. No.09/597,774, which is a continuation-in-part application of applicationSer. No. 09/250,280 which is a continuation-in-part application ofapplication Ser. No. 09/105,812, filed Jun. 26, 1998, and incorporatedherein in its entirety by reference thereto.

FIELD OF THE INVENTION

The present invention relates to, polynucleotides which encode plantpolypeptides and which exhibit senescence-induced expression, transgenicplants containing the polynucleotides in antisense orientation andmethods for controlling senescence in plants. More particularly, thepresent invention relates to plant lipase genes whose expression isinduced by the onset of senescence and the use of the lipase gene tocontrol senescence in plants.

DESCRIPTION OF THE PRIOR ART

Senescence is the terminal phase of biological development in the lifeof a plant. It presages death and occurs at various levels of biologicalorganization including the whole plant, organs, flowers and fruit,tissues and individual cells.

Cell membrane deterioration is an early and fundamental feature ofsenescence. Metabolism of lipids, in particular membrane lipids, is oneof several biochemical manifestations of cellular senescence. Rosepetals, for example, sustain an increase in acyl hydrolase activity assenescence progresses that is accompanied by a loss of membrane function(Borochov, et al., Plant Physiol., 1982, 69, 296-299). Cell membranedeterioration is an early and characteristic feature of senescenceengendering increased permeability, loss of ionic gradients anddecreased function of key membrane proteins such as ion pumps (Brown, etal., Plant Physiol.: A Treatise, Vol. X. Academic Press, 1991,pp.227-275). Much of this decline in membrane structural and functionalintegrity can be attributed to lipase-mediated phospholipid metabolism.Loss of lipid phosphate has been demonstrated for senescing flowerpetals, leaves, cotyledons and ripening fruit (Thompson, J. E.,Senescence and Aging in Plants, Academic Press, San Diego, 1988, pp.51-83), and this appears to give rise to major alterations in themolecular organization of the membrane bilayer with advancing senescencethat lead to impairment of cell function. In particular, studies with anumber of senescing plant tissues have provided evidence for lipid phaseseparations in membranes that appear to be attributable to anaccumulation of lipid metabolites in the membrane bilayer (McKersie andThompson, 1979, Biochim. Biophys. Acta, 508: 197-212; Chia, et al.,1981, Plant Physiol., 67:415-420). There is growing evidence that muchof the metabolism of lipids in senescing tissue is achieved throughsenescence-specific changes in gene expression (Buchanan-Wollaston, V.,J. Exp. Bot., 1997, 307:181-199).

The onset of senescence can be induced by different factors bothinternal and external. For example, ethylene plays a role in many plantsin a variety of plant processes such as seed germination, seedlingdevelopment, fruit ripening and flower senescence. Ethylene productionin plants can also be associated with trauma induced by mechanicalwounding, chemicals, stress (such as produced by temperature and wateramount variations), and by disease. Ethylene has been implicated in theregulation of leaf senescence in many plants, but evidence obtained withtransgenic plants and ethylene response mutants has indicated that,although ethylene has an effect on senescence, it is not an essentialregulator of the process. In many plants ethylene seems to have no rolein fruit ripening or senescence. For example in the ripening of fruitsof non-climacteric plants such as strawberry, in senescence of someflowers such as day lilies and in leaf senescence in some plants, suchas Arabidopsis, and in particular, in the monocots there is norequirement for ethylene signaling (Smart, C. M., 1994, New Phytology,126:419-448; Valpuesta, et al., 1995, Plant Mol. Biol., 28:575-582).

External factors that induce premature initiation of senescence includeenvironmental stresses such as temperature, drought, poor light ornutrient supply, as well as pathogen attack. As in the case of natural(age-related) senescence, environmental stress-induced senescence ischaracterized by a loss of cellular membrane integrity. Specifically,exposure to environmental stress induces electrolyte leakage reflectingmembrane damage (Sharom, et al., 1994, Plant Physiol., 105:305-308;Wright and Simon, 1973, J. Exp. Botany, 24:400-411; Wright, M., 1974,Planta,120:63-69; and Eze et al., 1986, Physiologia Plantarum,68:323-328), a decline in membrane phospholipid levels (Wright, M.,1974, Planta,120:63-69) and lipid phase transitions (Sharom, et al.,1994, Plant Physiol., 105:305-308), all of which can be attributed tothe action of lipase. Plant tissues exposed to environmental stress alsoproduce ethylene, commonly known as stress ethylene (Buchanan-Wollaston,V., 1997, J. Exp. Botany, 48:181-199; Wright, M., 1974,Planta,120:63-69). As noted above, ethylene is known to cause senescencein some plants.

Membrane deterioration leading to leakage is also a seminal feature ofseed aging, and there is evidence that this too reflectsdeesterification of fatty acids from membrane phospholipids (McKersie,B. D., Senarata, T., Walker, M. A., Kendall, E. J. and Hetherington, P.R. In: Senescence and Aging in Plants, Ed. L. D. Nooden and A. C.Leoopold, academic Press, 1988. PP 441-464).

Presently, there is no widely applicable method for controlling onset ofsenescence caused by either internal or external, e.g., environmentalstress, factors. At present, the technology for controlling senescenceand increasing the shelf-life of fresh, perishable plant produce, suchas fruits, flowers and vegetables relies primarily upon reducingethylene biosynthesis. For example, U.S. Pat. No. 5,824,875 disclosestransgenic geranium plants which exhibit prolonged shelf-life due toreduction in levels of ethylene resulting from the expression of one ofthree 1-amino-cyclopropane-1-carboxylate (ACC) synthase genes inantisense orientation. Consequently, this technology is applicable toonly a limited range of plants that are ethylene-sensitive.

The shelf-life of some fruits is also extended by reducing ethylenebiosynthesis, which causes ripening to occur more slowly. Sincesenescence of these fruits is induced after ripening, the effect ofreduced ethylene biosynthesis on shelf-life is indirect. Anotherapproach used to delay fruit ripening is by altering cellular levels ofpolygalacturonase, a cell-wall softening enzyme that is synthesizedduring the early stages of ripening. This approach is similar tocontrolling ethylene biosynthesis in that it, too, only indirectlyaffects senescence and again, is only applicable to a narrow range ofplants.

Thus, there is a need for a method of controlling senescence in plantswhich is applicable to a wide variety of plants. It is therefore ofinterest to develop senescence modulating technologies that areapplicable to all types of plants, regardless of ethylene sensitivity.

SUMMARY OF THE INVENTION

This invention is based on the discovery and cloning of a full lengthcDNA clone encoding a carnation senescence-induced lipase and afull-length cDNA clone encoding Arabidopdis thaliana senescence-inducedlipase. The nucleotide sequences and corresponding amino acid sequencesfor the senescence-induced lipase genes are disclosed herein. Thenucleotide sequence of the carnation senescence-induced lipase gene hasbeen successfully used as a heterologous probe to detect correspondinggenes or RNA transcripts in several plants that are similarly regulated.

The invention provides a method for genetic modification of plants tocontrol the onset of senescence, either age-related senescence orenvironmental stress-induced senescence. The senescence-induced lipasenucleotide sequences of the invention, fragments thereof, orcombinations of such fragments, are introduced into a plant cell inreverse orientation to inhibit expression of the endogenoussenescence-induced lipase gene, thereby reducing the level of endogenoussenescence-induced lipase and altering senescence in the transformedplant.

Using the methods of the invention, transgenic plants are generated andmonitored for growth and development. Plants or detached parts of plants(e.g., cuttings, flowers, vegetables, fruits, seeds or leaves)exhibiting prolonged life or shelf life with respect to plant growth,flowering, reduced fruit spoilage, reduced seed aging and/or reducedyellowing of leaves due to reduction in the level of senescence-inducedlipase are selected as desired products having improved propertiesincluding reduced leaf yellowing, reduced petal abscission, reducedfruit spoilage during shipping and storage. These superior plants arepropagated. Similarly, plants exhibiting increased resistance toenvironmental stress, e.g., decreased susceptibility to low temperature(chilling), drought, infection, etc., are selected as superior products.

In one aspect, the present invention is directed to an isolated DNAmolecule encoding senescence-induced lipase, wherein the DNA moleculehybridizes with SEQ ID NO:1, or a functional derivative of the isolatedDNA molecule which hybridizes with SEQ ID NO:1. In one embodiment of theinvention, the isolated DNA molecule has the nucleotide sequence of SEQID NO:1, i.e., 100% complementarity (sequence identity) to SEQ ID NO:1.In another embodiment of this aspect of the invention, the isolated DNAmolecule contains the nucleotide sequence of SEQ ID NO:4.

The invention is also directed to an isolated DNA molecule encodingsenescence-induced lipase, wherein the DNA molecule hybridizes with SEQID NO:18, or a functional derivative of the isolated DNA molecule whichhybridizes with SEQ ID NO:18. In one embodiment of this aspect of theinvention, the isolated DNA molecule has the nucleotide sequence of SEQID NO:18, i.e., 100% complementarity (sequence identity) to SEQ IDNO:18. In another embodiment of this aspect of the invention, theisolated DNA molecule contains the nucleotide sequence of SEQ ID NO:19.

In another embOdiment of the invention, there is provided an isolatedprotein encoded by a DNA molecule as described herein above, or afunctional derivative thereof. A preferred protein has the amino acidsequence of SEQ ID NO:2, or is a functional derivative thereof.

Also provided herein is an antisense oligonucleotide or polynucleotideencoding an RNA molecule which is complementary to at least a portion ofan RNA transcript of the DNA molecule described hereinabove, wherein theRNA molecule hybridizes with the RNA transcript such that expression ofendogenous senescence-induced lipase is altered. The antisenseoligonucleotide or polynucleotide can be full length or preferably hasabout six to about 100 nucleotides.

The antisense oligonucleotide or polynucleotide is substantiallycomplementary to a corresponding portion of one strand of a DNA moleculeencoding senescence-induced lipase, wherein the DNA molecule encodingsenescence-induced lipase hybridizes with SEQ ID NO:1, SEQ ID NO:18 orboth, or is substantially complementary to a corresponding portion of anRNA sequence encoded by the DNA molecule encoding senescence-inducedlipase. In one embodiment of the invention, the antisenseoligonucleotide or polynucleotide is substantially complementary to acorresponding portion of one strand of the nucleotide sequence SEQ IDNO:1,SEQ ID NO:18 or both or the RNA transcript encoded by SEQ ID NO:1.In another embodiment, the antisense oligonucleotide is substantiallycomplementary to a corresponding portion of about 100 to about 200nucleotides of the 5′ non-coding portion or 3′-end portion of one strandof a DNA molecule encoding senescence-induced lipase, wherein the DNAmolecule hybridizes with SEQ ID NO:1, SEQ ID NO:18 or both. In anotherembodiment, the antisense oligo- or polynucleotide is substantiallycomplementary to a corresponding portion of the open reading frame ofone strand of the nucleotide sequence SEQ ID NO:4 or the RNA transcriptencoded by SEQ ID NO:4.

The invention is further directed to a vector for transformation ofplant cells, comprising

(a) antisense nucleotide sequences substantially complementary to (1) acorresponding portion of one strand of a DNA molecule encodingsenescence-induced lipase, wherein the DNA molecule encodingsenescence-induced lipase hybridizes with SEQ ID NO:1,SEQ ID NO:18 orboth or (2) a corresponding portion of an RNA sequence encoded by theDNA molecule encoding senescence-induced lipase; and

(b) regulatory sequences operatively linked to the antisense nucleotidesequences such that the antisense nucleotide sequences are expressed ina plant cell into which it is transformed.

The regulatory sequences include a promoter functional in thetransformed plant cell, which promoter may be inducible or constitutive.Optionally, the regulatory sequences include a polyadenylation signal.

The invention also provides a plant cell transformed with the vector asdescribed above, a plantlet or mature plant generated from such a cell,or a plant part of such a plantlet or plant.

The present method is further directed to a method of producing a planthaving a reduced level of senescence-induced lipase compared to anunmodified plant, comprising:

(1) transforming a plant with a vector as described above;

(2) allowing the plant to grow to at least a plantlet stage;

(3) assaying the transformed plant or plantlet for alteredsenescence-induced lipase activity and/or altered senescence and/oraltered environmental stress-induced senescence and/or ethylene-inducedsenescence; and

(4) selecting and growing a plant having altered senescence-inducedlipase activity and/or altered senescence and/or altered environmentalstressed-induced senescence or ethylene-induced senescence compared toan nom-transformed plant.

A plant produced as above, or progeny, hybrids, clones or plant partspreferably exhibit reduced senescence-induced lipase expression anddelayed senescence and/or delayed stress-induced senescence orethylene-induced senescence.

This invention is further directed to a method of inhibiting expressionof endogenous senescence-induced lipase in a plant cell, said methodcomprising:

(1) integrating into the genome of a plant a vector comprising

-   -   (A) antisense nucleotide sequences complementary to (i) a        corresponding portion of one strand of a DNA molecule encoding        endogenous senescence-induced lipase, wherein the DNA molecule        encoding the endogenous senescence-induced lipase hybridizes        with SEQ ID NO:1, SEQ ID NO:18 or both, or (ii) a corresponding        portion of an RNA sequence encoded by the endogenous        senescence-induced lipase gene; and    -   (B) regulatory sequences operatively linked to the antisense        nucleotide sequences such that the antisense nucleotide        sequences are expressed; and

(2) growing said plant, whereby said antisense nucleotide sequences aretranscribed and the transcript binds to said endogenous RNA wherebyexpression of said senescence-induced lipase gene is inhibited.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the derived amino acid sequence (SEQ ID NO:2) encoded bythe senescence-induced lipase cDNA clone (SEQ ID NO:1) obtained from acarnation flower cDNA library. Consensus motifs within the amino acidsequence are as follows: single underline, amidation site; dottedunderline, protein kinase C phosphorylation site; double underline,N-myristoylation site; box border, cAMP phosphorylation site; shadowbox, casein kinase II phosphorylation site; cross-hatched box, consensussequence of lipase family; and dotted box, N-glycosylation site.

FIG. 2 depicts the derived full length carnation petalsenescence-induced lipase amino acid sequence (SEQ ID NO:2) in alignmentwith partial sequences of lipase-like proteins. Carlip, full lengthsequence of carnation petal senescence-induced lipase (SEQ ID NO:11);arlip, partial sequence of lipase-like protein from Arabidopsis thaliana(Gen Bank Accession No. AL021710) (SEQ ID NO:12); ipolip, partialsequence of a lipase-like sequence from Ipomea (Gen Bank Accession No.U55867) (SEQ ID NO:13); arlipi, partial sequence of lipase-like proteinfrom Arabidopsis thaliana (Gen Bank Accession No. U93215) (SEQ IDNO:14). Identical amino acids among three or four of the sequences areboxed.

FIG. 3 shows a Western blot analysis of the fusion protein expressionproduct obtained from carnation lipase cDNA expressed in E. coli. TheWestern blot was probed with antibodies to the senescence-induced lipaseprotein. Lane 1, maltose binding protein; lane 2, fusion proteinconsisting of carnation lipase fused through a proteolytic (Factor Xa)cleavage site to maltose binding protein cDNA; lane 3, fusion proteinpartially cleaved with Factor Xa into free lipase protein (50.2 kDa)andfree maltose-binding protein.

FIG. 4 is a Northern blot analysis of RNA isolated from carnation flowerpetals at different stages of development. FIG. 4A is the ethidiumbromide stained gel of total RNA. Each lane contained 10 μg RNA. FIG. 4Bis an autoradiograph of the Northern blot probed with ³²P-dCTP-labelledfull length carnation senescence-induced lipase cDNA.

FIG. 5 is an in situ demonstration of lipolytic acyl hydrolase, i.e.,lipase activity of the protein product obtained by over expression ofthe carnation senescence-induced lipase cDNA in E. coli. mal, E. colicells containing maltose binding protein alone in a basal salt medium;mLip, E. coli cells containing the fusion protein consisting of thecarnation senescence-induced lipase fused with maltose binding proteinin basal salt medium; 40 mal/40 mLip, E. coil cells containing maltosebinding protein alone [mal] or the lipase-maltose binding protein fusionproduct [mLip] in basal salt medium supplemented with Tween 40; 60mal/60 mLip, E. coli cells containing maltose binding protein alone[mal] or the lipase-maltose binding protein fusion product [mLip] inbasal salt medium supplemented with Tween 60.

FIG. 6A illustrates a restriction enzyme map of the open reading frameof the carnation senescence-induced lipase. The numbers refer tonucleotides in the open reading frame.

FIG. 6B is a Southern blot analysis of carnation genomic DNA digestedwith various restriction enzymes and probed with carnationsenescence-induced lipase cDNA.

FIG. 7 is the nucleotide sequence of the carnation senescence-inducedlipase cDNA clone (SEQ ID NO:1). Solid underlining, non-coding sequenceof the senescence-induced lipase cDNA; non-underlined sequenced is theopen reading frame.

FIG. 8 is the amino acid sequence of the carnation senescence-inducedlipase cDNA (SEQ ID NO:2).

FIG. 9A is a Northern blot analysis showing the expression of thecarnation lipase in stage II petals that have been exposed to 0.5 ppmethylene for 15 hours. FIG. 9A is an ethidium bromide stained gelshowing that each of the lanes was loaded with a constant amount ofcarnation RNA (petals: lanes 1 and 2; leaves: lanes 3 and 4; +, ethylenetreated; −, untreated). FIG. 9B is an autoradiogram of a Northern blotof the gel in FIG. 9A probed with labelled full length carnation petalsenescence-induced lipase cDNA.

FIG. 10 is a partial nucleotide sequence of tomato leaf genomicsenescence-induced lipase (SEQ ID NO:6) and the corresponding deducedamino acid sequence (SEQ ID NO:17).

The conserved lipase consensus motif is shaded; the sequences of theprimers used to generate the genomic fragment are each underlined.

FIG. 11 is a bar graph showing the effects of chilling on membraneleakiness. Tomato plants were chilled at 8° for 48 hours and thenrewarmed to room temperature. Diffusate leakage (μMhos) from leaf diskswas measured for control plants, ,which had not been chilled, and forchilled plants for 6 and 24 hour periods.

FIG. 12 is a Northern blot analysis of tomato leaf RNA isolated fromplants that had been chilled at 8° C. for 48 hours and rewarmed toambient temperature for 24 hours. FIG. 12A is the ethidium bromidestained gel of total leaf RNA. FIG. 12B is an autoradiograph of theNorthern blot probed with ³²P-dCTP-labelled full length carnationsenescence-induced lipase cDNA.

FIG. 13 is a partial nucleotide sequence (SEQ ID NO:15) andcorresponding deduced amino acid sequence of an Arabidopsis EST (GenBankAcc#: N38227) (SEQ ID NO:16) that is 55.5% identical over a 64 aminoacid region with the carnation senescence-induced lipase. The conservedlipase consensus motif is shaded.

FIG. 14 is the nucleotide (top) (SEQ ID No:18) and derived amino acidsequence (bottom) (SEQ ID NO:19) of the full-length Arabidopsissenescence-induced lipase gene.

FIG. 15 is a Northern blot of total RNA isolated from leaves ofArabidopsis plants at various stages (lane 1, two week-old plants; lane2, three week-old plants; lane 3, four week-old plants; lane 4, fiveweek-old plants; lane 5, six week-old plants) probed with³²P-dCTP-labelled full-length Arabidopsis senescence-induced lipase. Theautoradiograph is at the top (15A) and the ethidium bromide stained gelbelow(15B).

FIG. 16 is a Northern blot of total RNA isolated from leaves of threeweek-old Arabidopsis plants treated with 50 μM ethephon (a source ofethylene) and probed with ³²P-dCTP-labelled full-length Arabidopsissenescence-induced lipase. The autoradiograph is at the top (16A) andthe ethidium bromide stained gel below(16B).

FIG. 17 is a photograph of 4.6 week-old Arabidopsis wild-type plants(left) and transgenic plants (right) expressing the full-lengthArabidopsis senescence-induced lipase gene in antisense orientationshowing increased leaf size in the transgenic plants.

FIG. 18 is a photograph of 6.3 week-old Arabidopsis wild-type plants(left) and transgenic plants (right) expressing the full-lengthArabidopsis senescence-induced lipase gene in antisense orientationshowing increased leaf size and delayed leaf senescence in thetransgenic plants.

FIG. 19 is a photograph of 7 week-old Arabidopsis wild-type plants(left) and transgenic plants (right) expressing the full-lengthArabidopsis senescence-induced lipase gene in antisense orientationshowing increased leaf size in the transgenic plants.

FIG. 20 is a graph showing the increase in seed yield in three T₁transgenic Arabidopsis plant lines expressing the senescence-inducedlipase gene in antisense orientation. Seed yied is expressed as volumeof seed. SE for n=30 is shown for wild-type plants.

FIG. 21 is a Western blot of total protein isolated from leaves of fourweek-old Arabidopsis wild-type plants and corresponding transgenicplants expressing the full-length senescence-induced lipase gene inantisense orientation. (Lanes 1 and 2 were loaded with 9 μg of protein,and lanes 3 and 4 were loaded with 18 μg of protein). The blot wasprobed with antibody raised against the Arabidopsis senescence-inducedlipase protein. The expression of the senescence-induced lipase isreduced in all transgenic plants.

DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions are provided for altering the expression ofsenescence-induced lipase gene(s)in plant cells. Alteration ofexpression of the senescence-induced lipase gene(s) in plants results indelayed onset of senescence and improved resistance to environmentalstress, thus extending the plant shelf-life and/or growth period.

A full length cDNA sequence encoding a carnation lipase gene exhibitingsenescence-induced expression has been isolated from a cDNA library madefrom RNA of senescing petals of carnation (Dianthuscaryophyllus)flbwers. Polynucleotide probes corresponding to selectedregions of the isolated carnation flower lipase cDNA sequence as well asthe full length carnation lipase cDNA were used to determine thepresence of mRNA encoding the lipase gene in senescing carnation leaves,ripening tomato fruit and senescing green bean leaves, as well asenvironmentally stressed (chilled) tomato leaves. Primers designed fromthe carnation lipase cDNA were used to generate a polymerase chainreaction (PCR) product using tomato leaf genomic DNA as template. ThePCR product contains a partial open reading frame which encodes apartial protein sequence including the conserved lipase consensus motif,ITFTGHSLGA (SEQ ID NO:3). The tomato nucleotide sequence has 53,4%sequence identity with the carnation senescence-induced lipase sequenceand 43.5% identity with Arabidopsis lipase sequence. The Arabidopsislipase sequence has 44.3% identity with the carnation nucleotidesequence.

The carnation senescence-induced lipase gene of the present inventionwas isolated by screening a cDNA expression library prepared fromsenescing carnation petals with antibodies raised against cytosoliclipid-protein particles, a source of the carnation lipase. A positivefull-length cDNA clone corresponding to the carnation senescence-inducedlipase gene was obtained and sequenced. The nucleotide sequence of thesenescence-induced lipase cDNA clone is shown in SEQ ID NO:1. The cDNAclone encodes a 447 amino acid polypeptide (SEQ ID NO: 2) having acalculated molecular mass of 50.2 kDa. Expression of the cDNA clone inE. coli yielded a protein of the expected molecular weight that exhibitsacyl hydrolase activity, i.e., the expressed protein hydrolyzesp-nitrophenylpalmitate, phospholipid and triacylglycerol. Based on theexpression pattern of the enzyme in developing carnation flowers and theactivity of the protein, it is involved in senescence.

An Arabidopsis senescence-induced lipase gene of the present inventionwas also isolated by PCR using a senescing Arabidopsis leaf cDNA libraryas template in the reaction. The nucleotide and derived amino acidsequence of the Arabidopsis senescence-induced lipase gene is shown inFIG. 14 (SEQ ID NO:18) Based on the expression pattern of the lipasegene in developing plants, it is involved in senescence.

Northern blots of carnation petal total RNA probed with the full lengthcarnation cDNA show that the expression of the senescence-induced lipasegene is significantly induced just prior to the onset of naturalsenescence (FIG. 4). Northern blot analyses also demonstrate that thesenescence-induced lipase gene is induced by environmental stressconditions, e.g., chilling (FIG. 12) and .ethylene (FIGS. 4 and 9),which is known to be produced in response to environmental stress. TheNorthern blot analyses show that the presence of carnationsenescence-induced lipase mRNA is significantly higher in senescing(developmental stage IV) than in young stage I, II and III carnationpetals. Furthermore, ethylene-stimulated stage II flowers also showhigher senescence-induced lipase gene expression. Similarly, plants thathave been exposed to chilling temperatures and returned to ambienttemperature also show induced expression of the senescence-inducedlipase gene coincident with the development of chilling injury symptoms(e.g., leakiness) (FIGS. 11 and 12).

Expression of the Arabidopsis senescence-induced lipase gene issimilarly regulated. Northern blot analysis of total RNA from leaves ofArabidopsis plants at various stages of development show that the lipasegene is upregulated coincident with the onset of leaf senescence.(FIG.15) Also, like the carnation senescence-induced lipase gene, theArabidopsis senescence-induced lipase gene is upregulated by treatmentwith ethylene, a plant hormone that induces leaf senescence. (FIG. 16)

The overall pattern of gene expression in various plants, e.g.,carnation, green beans, tomato, Arabidopsis, and various plant tissues,e.g., leaves, fruit and flowers, demonstrates that the lipase genes ofthe invention are involved in the initiation of senescence in theseplants and plant tissues. Thus, it is expected that by substantiallyrepressing or altering the expression of the senescence-induced lipasegenes in plant tissues, senescence, deterioration and spoilage can bedelayed, increasing the shelf-life of perishable fruits, flowers andvegetables. This can be achieved by producing transgenic plants in whichthe lipase cDNA or an oligonucleotide fragment thereof is expressed inthe antisense configuration in fruits, flowers, vegetable, agronomiccrop plants and forest species, preferably using a constitutive promotersuch as the CaMV 35S promoter, or using a tissue-specific orsenescence-inducible promoter.

The carnation senescence-induced lipase gene is a single copy gene.Southern blot analysis of carnation genomic DNA cut with variousrestriction enzymes that do not recognize sequences within the openreading frame of the senescence-induced lipase cDNA was carried out. Therestriction enzyme-digested genomic DNA was probed with³²P-dCTP-labelled full length cDNA (SEQ ID NO:1). Under high stringencyhybridization conditions, only one restriction fragment hybridizes tothe cDNA clone (68° C. for both hybridization and washing; washingbuffer :0.2%×SSC, 0.1% SDS). Thus, the carnation senescence-inducedlipase gene is a single copy gene (FIG. 6). The fact that this gene isnot a member of a multigene family in carnations strongly suggests thatit is a single copy gene in other plants.

Knowledge of the complete nucleotide sequence of the carnationsenescence-induced lipase gene orArabidopsis senescence-induced lipasegene is sufficient for the isolation of the senescence-induced lipasegene(s) from various other plant species. Indeed, as demonstratedherein, oligonucleotide primers based on the carnation cDNA sequencehave been successfully used to generate tomato leaf senescence-inducedlipase gene fragments by polymerase chain reactions using tomato leafgenomic DNA as template.

The cloned senescence-induced lipase gene(s) or fragment(s) thereof,alone or in combination, when introduced in reverse orientation(antisense) under control of a constitutive promoter, such as the figwart mosaic virus 35S promoter, the cauliflower mosaic virus promoterCaMV35S or the MAS promoter, can be used to genetically modify plantsand alter senescence in the modified plants. Selected antisensesequences from other plants which share sufficient sequence identitywith the carnation senescence-induced lipase gene can be used to achievesimilar genetic modification. One result of the genetic modification isa reduction in the amount of endogenous translatable senescence-inducedlipase-encoding mRNA. Consequently, the amount of senescence-inducedlipase produced in the plant cells is reduced, thereby reducing theamount of cell membrane damage and cell leakage, e.g., reduced leaf,fruit and/or flower senescence and spoilage, due to aging orenvironmental stress.

For example, Arabidopsis plants transformed with vectors that expressthe full-length Arabidopsis senescence-induced lipase gene in antisenseorientation, under regulation of double 35S promoter exhibit larger leafsize and overall larger plant growth as compared to wild-type plants asshown in FIGS. 17 and 18. These plants also demonstrate delayed leafsenescence, as shown in FIG. 19.

The effect of reduced expression of the senescence-induced lipase genebrought about by expressing the full-length lipase gene in antisenseorientation in transgenic Arabidopsis plants is also seen as an increasein seed yield in the transformed plants. Arabidopsis plant linesexpressing the full-length senescence-induced lipase gene produce up toabout two to three times more seed than wild type plants. (FIG. 20)

That the effects observed in transgenic plants on biomass, leafsenescence and seed yield are due to a decrease in senescence-inducedlipase in these plants is shown in FIG. 21. The transgenic plants of theinvention exhibit significantly reduced expression of senescence-inducedlipase in comparison to wild-type plants.

Thus, the methods and sequences of the present invention can be used todelay plant spoilage, including leaf or fruit spoilage, as well as toincrease plant biomass and seed yield, and in general, alter senesencein plants.

The isolated nucleotide sequences of this invention can be used toisolate substantially complementary senescence-induced lipase nucleotidesequence from other plants or organisms. These sequences can, in turn,be used to transform plants and thereby alter senescence of thetransformed plants in the same manner as shown with the use of theisolated nucleotide sequences shown herein.

The genetic modifications observed with transformation of plants withsenescence-induced lipase, functional fragments thereof or combinationsthereof can effect a permanent change in levels of senescence-inducedlipase in the plant and be propagated in offspring plants by selfing orother reproductive schemes. The genetically altered plant is used toproduce a new line of plants wherein the alteration is stablytransmitted from generation to generation. The present inventionprovides for the first time the appropriate DNA sequences which may beused to achieve a stable genetic modification of senescence in a widerange of different plants.

For the identification and isolation of the senescence-induced lipasegene, in general, preparation of plasmid DNA, restriction enzymedigestion, agarose gel electrophoresis of DNA, polyacrylamide gelelectrophoresis of protein, Southern blots, Northern blots, DNA ligationand bacterial transformation were carried out using conventional methodswell-known in the art. See, for example, Sambrook, J. et al., MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, ColdSpring Harbor, N.Y., 1989. Techniques of nucleic acid hybridization aredisclosed by Sambrook (Supra).

As used herein, the term “plant” refers to either a whole plant, a plantpart, a plant cell or a group of plant cells. The type of plant whichcan be used in the method of the invention is not limited and includes,for example, ethylene-sensitive and ethylene-insensitive plants; fruitbearing plants such as apricots, apples, oranges, bananas, grapefruit,pears, tomatoes, strawberries, avocados, etc.; vegetables such ascarrots, peas, lettuce, cabbage, turnips, potatoes, broccoli, asparagus,etc.; flowers such as carnations, roses, mums, etc.; and in general, anyplant that can take up and express the DNA molecules of the presentinvention. It may include plants of a variety of ploidy levels,including haploid, diploid, tetraploid and polyploid.

A transgenic plant is defined herein as a plant which is geneticallymodified in some way, including but not limited to a plant which hasincorporated heterologous or homologous senescence-induced lipase DNA ormodified DNA or some portion of heterologous senescence-induced lipaseDNA or homologous senescence-induced lipase DNA into its genome. Thealtered genetic material may encode a protein, comprise a regulatory orcontrol sequence, or may be or include an antisense sequence or encodean antisense RNA which is antisense to the endogenous senescence-inducedlipase DNA or mRNA sequence or portion thereof of the plant. A“transgene” or “transgenic sequence” is defined as a foreign gene orpartial sequence which has been incorporated into a transgenic plant.

The term “hybridization” as used herein is generally used to meanhybridization of nucleic acids at appropriate conditions of stringencyas would be readily evident to those skilled in the art depending uponthe nature of the probe sequence and target sequences. Conditions ofhybridization and washing are well known in the art, and the adjustmentof conditions depending upon the desired stringency by varyingincubation time, temperature and/or ionic strength of the solution arereadily accomplished. See, for example, Sambrook, J. et al., MolecularCloning: A Laboratory Manual, 2nd edition, Cold spring harbor Press,Cold Spring harbor, N.Y., 1989. The choice of conditions is dictated bythe length of the sequences being hybridized, in particular, the lengthof the probe sequence, the relative G-C content of the nucleic acids andthe amount of mismatches to be permitted. Low stringency conditions arepreferred when partial hybridization between strands that have lesserdegrees of complementarity is desired. When perfect or near perfectcomplementarity is desired, high stringency conditions are preferred.For typical high stringency conditions, the hybridization solutioncontains 6×S.S.C., 0.01 M EDTA, 1×Denhardt's solution and 0.5% SIDS_(—)Hybridization is carried out at about 68° C. for about 3 to 4 hours forfragments of cloned DNA and for about 12 to about 16 hours for totaleukaryotic DNA. For lower stringencies the temperature of hybridizationis reduced to about 12° C. below the melting temperature (T_(M)) of theduplex. The T_(M) is known to be a function of the G-C content andduplex length as well as the ionic strength of the solution.

As used herein, the term “substantial sequence identity” or “substantialhomology” is used to^(.)indicate that a nucleotide sequence or an aminoacid sequence exhibits. substantial structural or functional equivalencewith another nucleotide or amino acid sequence. Any structural orfunctional differences between sequences having substantial sequenceidentity or substantial homology will be de minimis; that is, they willnot affect the ability of the sequence to function as indicated in thedesired application. Differences may be due to inherent variations incodon usage among different species, for example. Structural differencesare considered de minimis if there is a significant amount of sequenceoverlap or similarity between two or more different sequences or if thedifferent sequences exhibit similar physical characteristics even if thesequences differ in length or structure-. Such characteristics includefor example, ability to hybridize under defined conditions, or in thecase of proteins, immunological crossreactivity, similar enzymaticactivity, etc.

Additionally, two nucleotide sequences are “substantially complementary”if the sequences have at least about 40 percent, more preferably, atleast about 60 percent and most preferably about 90 percent sequencesimilarity between them. Two amino acid sequences are substantiallyhomologous if they have at least 40%, preferably 70% similarity betweenthe active portions of the polypeptides.

As used herein, the phrase “hybridizes to a corresponding portion” of aDNA or RNA molecule means that the molecule that hybridizes, e.g.,oligonucleotide, polynucleotide, or any nucleotide sequence (in sense orantisense orientation) recognizes and hybridizes to a sequence inanother nucleic acid molecule that is of approximately the same size andhas enough sequence similarity thereto to effect hybridization underappropriate conditions. For example, a 100 nucleotide long antisensemolecule from the 3′ coding or non-coding region of carnation lipasewill recognize and hybridize to an approximately 100 nucleotide portionof a nucleotide sequence within the 3′ coding or non-coding region,respectively of the Arabidopsis senescence-induced lipase gene or anyother plant senescence-induced lipase gene so long as there is about 70%or more sequence similarity between the two sequences. It is to beunderstood that the size of the “corresponding portion” will allow forsome mismatches in hybridization such that the “corresponding portion”may be smaller or larger than the molecule which hybridizes to it, forexample 20-30% larger or smaller, preferably no more than about 12-15%larger or smaller.

The term “functional derivative” of a nucleic acid (or poly- oroligonucleotide) is used herein to mean a fragment, variant, homolog, oranalog of the gene or nucleotide sequence encoding senescence-inducedlipase. A functional derivative may retain at least a portion of thefunction of the senescence-induced lipase encoding DNA which permits itsutility in accordance with the invention. Such function may include theability to hybridize with native carnation senescence-induced lipase orsubstantially homologous DNA from another plant which encodessenescence-induced lipase or with an mRNA transcript thereof, or, inantisense orientation, to inhibit the transcription and/or translationof plant senescence-induced lipase mRNA, or the like.

A “fragment” of the gene or DNA sequence refers to any subset of themolecule, e.g., a shorter polynucleotide or oligonucleotide. A “variant”refers to a molecule substantially similar to either the entire gene ora fragment thereof, such as a nucleotide substitution variant having oneor more substituted nucleotides, but which maintains the ability tohybridize with the particular gene or to encode mRNA transcript whichhybridizes with the native DNA. A “homolog” refers to a fragment orvariant sequence from a different plant genus or species. An “analog”refers to a non-natural molecule substantially similar to or functioningin relation to either the entire molecule, a variant or a fragmentthereof.

By “altered expression” or “modified expression” of a gene, e.g., thesenescence-induced lipase gene, is meant any process or result wherebythe normal expression of the gene, for example, that expressionoccurring in an unmodified carnation or other plant, is changed in someway. As intended herein, alteration in gene expression is complete orpartial reduction in the expression of the senescence-induced lipasegene, but may also include a change in the timing of expression, oranother state wherein the expression of the senescence-induced lipasegene differs from that which would be most likely to occur naturally inan unmodified plant or cultivar. A preferred alteration is one whichresults in reduction of senescence-induced lipase production by theplant compared to production in an unmodified plant.

In producing a genetically altered plant in accordance with thisinvention, it is preferred to select individual plantlets or plants bythe desired trait, generally reduced senescence-induced lipaseexpression or production. Expression of senescence-induced lipase can bequantitated, for example in a conventional immunoassay method using aspecific antibody as described herein. Also, senescence-induced lipaseenzymatic activity can be measured using biochemical methods asdescribed herein.

In order for a newly inserted gene or DNA sequence to be expressed,resulting in production of the protein which it encodes, or in the caseof antisense DNA, to be transcribed, resulting in an antisense RNAmolecule, the proper regulatory elements should be present in properlocation and orientation with respect to the gene or DNA sequence. Theregulatory regions may include a promoter, a 5′-non-ttanslated leadersequence and a 3′-polyadenylation sequence as well as enhancers andother regulatory sequences.

Promoter regulatory elements that are useful in combination with thesenescence-induced lipase gene to generate sense or antisensetranscripts of the gene include any plant promoter in general, and moreparticularly, a constitutive promoter such as the fig wart mosaic virus35S promoter, double 35S promoter, the cauliflower mosaic viruspromoter, CaMV35S promoter, or the MAS promoter, or a tissue-specific orsenescence-induced promoter, such as the carnation petal GST1 promoteror the Arabidopsis SAG12 promoter (See, for example, J. C. Palaqui etal., Plant Physiol., 112:1447-1456 (1996); Morton et al., MolecularBreeding, 1:123-132 (1995); Fobert et al., Plant Journal, 6:567-577(1994); and Gan et al., Plant Physiol., 113:313 (1997), incorporatedherein by reference). Preferably, the promoter used in the presentinvention is a constitutive promoter.

Expression levels from a promoter which is useful for the presentinvention can be tested using conventional expression systems, forexample by measuring levels of a reporter gene product, e.g., protein ormRNA in extracts of the leaves, flowers, fruit or other tissues of atransgenic plant into which the promoter/reporter have been introduced.

The present invention provides antisense oligonucleotides andpolynucleotides complementary to the gene encoding carnationsenescence-induced lipase, complementary to the gene encodingArabidopsis senescence-induced lipase or complementary to a gene or genefragment from another plant, which hybridizes with the carnation orArabidopsis senescence-induced lipase gene under low to high stringencyconditions. Such antisense oligonucleotides should be at least about sixnucleotides in length to provide minimal specificity of hybridizationand may be complementary to one strand of DNA or mRNA encodingsenescence-induced lipase or a portion thereof, or to flanking sequencesin genomic DNA which are involved in regulating senescence-inducedlipase gene expression. The antisense oligonucleotide may be as large as100 nucleotides and may extend in length up to and beyond the fullcoding sequence for which it is antisense. The antisenseoligonucleotides can be DNA or RNA or chimeric mixtures of DNA and RNAor derivatives or modified versions thereof, single stranded or doublestranded.

The action of the antisense oligonucleotide may result in alteration,primarily inhibition, of senescence-induced lipase gene expression incells. For a general discussion of antisense see: Alberts, et al.,Molecular Biology of the Cell, 2nd ed., Garland Publishing, Inc. NewYork, N.Y. (1989, in particular pages 195-196, incorporated herein byreference).

The antisense oligonucleotide may be complementary to any portion of thesenescence-induced lipase gene. In one embodiment, the antisenseoligonucleotide may be between 6 and 100 nucleotides in length, and maybe complementary to the 5′-non-coding sequence or 3′end of thesenescence-induced lipase sequence, for example. Antisenseoligonucleotides primarily complementary to 5′-non-coding sequences areknown to be effective inhibitors of expression of genes encodingtranscription factors. Branch, M. A., Molec. Cell Biol., 13:4284-4290(1993).

Preferred antisense oligonucleotides are substantially complementary toa corresponding portion of the mRNA encoding senescence-induced lipase.For example, introduction of the full length cDNA clone encodingsenescence-induced lipase in an antisense orientation into a plant isexpected to result in successful altered senescence-induced lipase geneexpression. Moreover, introduction of partial sequences, targeted tospecific portions of the senescence-induced lipase gene, can be equallyeffective.

The minimal amount of homology required by the present invention is thatsufficient to result in sufficient complementarity to providerecognition of the specific target RNA or DNA and inhibition orreduction of its translation or function while not affecting function ofother RNA or DNA molecules and the expression of other genes. While theantisense oligonucleotides of the invention comprise sequencescomplementary to at least a portion of an RNA transcript of thesenescence-induced lipase gene, absolute complementarity, althoughpreferred is not required. The ability to hybridize may depend on thelength of the antisense oligonucleotide and the degree ofcomplementarity. Generally, the longer the hybridizing nucleic acid, themore base mismatches with the senescence-induced lipase target sequenceit may contain and still form a stable duplex. One skilled in the artcan ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting temperature of the hybridizedcomplex, for example.

The antisense RNA oligonucleotides may be generated intracellularly bytranscription from exogenously introduced nucleic acid sequences. Theantisense molecule may be delivered to a cell by transformation ortransfection or infection with a vector, such as a plasmid or virus intowhich is incorporated DNA encoding the antisense senescence-inducedlipase sequence operably linked to appropriate regulatory elements,including a promoter. Within the cell the exogenous DNA sequence isexpressed, producing an antisense RNA of the senescence-induced lipasegene.

Vectors can be plasmids, preferably, or may be viral or other vectorsknown in the art to replicate and express genes encoded thereon in plantcells or bacterial cells. The vector becomes chromosomally integratedsuch that it can be transcribed to produce the desired antisensesenescence-induced lipase RNA. Such plasmid or viral vectors can beconstructed by recombinant DNA technology methods that are standard inthe art. For example, the vector may be a plasmid vector containing areplication system functional in a prokaryotic host and an antisenseoligonucleotide or polynucleotide according to the invention.Alternatively, the vector may be a plasmid containing a replicationsystem functional in Agrobacterium and an antisense oligonucleotide orpolynucleotide according to the invention. Plasmids that are capable ofreplicating in Agrobacterium are well known in the art. See, Miki,etal., Procedures for Introducing Foreign DNA Into Plants, Methods inPlant Molecular Biology and Biotechnology, Eds. B. R. Glick and J. E.Thompson. CRC Press (1993), PP. 67-83.

The carnation lipase gene was cloned in the antisense orientation into aplasmid vector in the following manner. The pCD plasmid, which isconstructed from a pUC18 backbone and contains the 35S promoter fromcauliflower mosaic virus (CaMV) followed by a multiple cloning site andan octapine synthase termination sequence was used for cloning thecarnation lipase gene. The pCd-lipase (antisense) plasmid wasconstructed by subcloning the full length carnation lipase gene in theantisense orientation into a Hind3 site and EcoRl site of pCd.Similarly, a pCDΔ35S-GST1-lipase (antisense) plasmid was constructed byfirst subcloning a PCR amplified fragment (−703 to +19 bp) of thecarnation Glutathione S Transferase 1 (GST1) promoter into the BamHl andSal1 sites of the pCd vector. The full length carnation lipase gene wasthen subcloned in the antisense orientation into the Hind3 and EcoR1sites of the construct. Another plasmid, pGdΔ35S-GST1-GUS plasmid, wasconstructed by first subcloning a PCR-amplified fragment (−703 to +19bp) of the carnation Glutathione S-Transferase 1 (GST1) promoter intothe BamHl and Sal1 sites of the pCd vector. The reporter genebeta-glucuronidase (GUS) was then subcloned into the Sal1 and EcoRIsites of the construct. The pCd-35S²-lipase (antisense) plasmid wasconstructed by first subcloning a double 35S promoter (containing twocopies of the CaMV 35S promoter in tandem) into the Sma1 and Hind3 sitesof the pCd vector. The full length carnation lipase gene was thensubcloned in the antisense orientation into the Hind3 and EcoR1 sites ofthe construct.

An oligonucleotide, preferably between about 6 and about 100 nucleotidesin length and complementary to the target sequence of senescence-inducedlipase, may be prepared by recombinant nucleotide technologies or may besynthesized from mononucleotides or shorter oligonucleotides, forexample. Automated synthesizers are applicable to chemical synthesis ofthe oligo- and polynucleotides of the invention. Procedures forconstructing recombinant nucleotide molecules in accordance with thepresent invention are disclosed in Sambrook, et al., In: MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1989), which is incorporated herein in itsentirety. Oligonucleotides which encode antisense RNA complementary tosenescence-induced lipase sequence can be prepared using procedures wellknown to those in the art. Details concerning such procedures areprovided in Maniatis, T. et al., Molecular mechanisms in the Control ofGene expression, eds., Nierlich, et al., eds., Acad. Press, N.Y. (1976).

In an alternative embodiment of the invention, inhibition of expressionof endogenous plant senescence-induced lipase is the result ofco-suppression through over-expression of an exogenoussenescence-induced lipase gene or gene fragment introduced into theplant cell. In this embodiment of the invention, a vector encodingsenescence-induced lipase in the sense orientation is introduced intothe cells in the same manner as described herein for antisensemolecules. Preferably, the senescence-induced lipase is operativelylinked to a strong constitutive promoter, such as for example the figwart mosaic virus promoter or CaMV35S.

Transgenic plants made in accordance with the present invention may beprepared by DNA transformation using any method of plant transformationknown in the art. Plant transformation methods include directco-cultivation of plants, tissues or cells with Agrobacteriumtumerfaciens or direct infection (Miki, et al., Meth. in Plant Mol.Biol. and

Biotechnology, (1993), p. 67-88); direct gene transfer into protoplastsor protoplast uptake (Paszkowski, et al., EMBO J., 12:2717 (1984);electroporation (Fromm, et al., Nature, 319:719 (1986); particlebombardment (Klein et al., BioTechnology, 6:559-563 (1988); injectioninto meristematic tissues of seedlings and plants (De LaPena, et al.,Nature, 325:274-276 (1987); injection, into protoplasts of culturedcells and tissues (Reich, et al., BioTechnology, 4:1001-1004 (1986)).

Generally a complete plant is obtained from the transformation process.Plants are regenerated from protoplasts, callus, tissue parts orexplants, etc. Plant parts obtained from the regenerated plants in whichthe expression of senescence-induced lipase is altered, such as leaves,flowers, fruit, seeds and the like are included in the definition of“plant” as used herein. Progeny, variants and mutants of the regeneratedplants are also included in the definition of “plant.”

The present invention also provides carnation or Arabidopsissenescence-induced lipase protein encoded by the cDNA molecules of theinvention and proteins which cross-react with antibody to the carnationor Arabidopsis protein. Such proteins have the amino acid sequence setforth in SEQ ID No:2, shown in FIG. 1, share cross reactivity withantibodies to the protein set forth in SEQ ID NO:2, have the amino acidsequence set forth in SEQ ID NO:19 (shown in FIG. 14) or. share crossreactivity with antibodies to the protein set forth in SEQ ID NO:19.

The carnation or Arabidopsis senescence-induced lipase protein orfunctional derivatives thereof are preferably produced by recombinanttechnologies, optionally in combination with chemical synthesis methods.In one embodiment of the invention the senescence-induced lipase isexpressed as a fusion protein consisting of the senescence-inducedlipase fused with maltose binding protein. Expression of a cloneencoding the recombinant fusion protein yields a fusion protein of theexpected molecular weight that hydrolyzes p-nitrophenylpalmitate,phospholipid and triacylglycerol, which is an indicator of lipaseactivity. The recombinant senescence-induced lipase protein shows apredoniinant band in Western blot analyses after immunoblotting withantibody to carnation senescence-induced lipase. The freesenescence-induced lipase (50.2 Kda), which is released by treatment ofthe fusion protein with the protease, factor Xa, also reacts with thesenescence-induced lipase antibody in Western blot analysis (FIG. 3). Amotif search of the senescence-induced lipase amino acid sequence showsthe presence of a potential N-myristoylation site (FIG. 1) for thecovalent attachment of myristate via an amide linkage (See Johnson, etal., Ann. Rev. Biochem., 63: 869-914 (1994); Towler, et al., Ann. Rev.Biochem., 57:67-99 (1988); and R.J.A. Grand, Biochem. J., 258:625-638(1989). The protein motif search also showed that the carnationsenescence-induced lipase contains a sequence, ITFAGHSLGA, (SEQ ID NO:4)which is the conserved lipase consensus sequence (Table 1). Theconserved lipase consensus sequence from a variety of plants is shown inthe table below.

TABLE 1 Plant Species conserved Lipase Sequence CarnationI T F A G H S L G A (SEQ ID NO: 4) Tomato I T F T G H S L G A(SEQ ID NO: 3) Arabidopsis I T T C G H S L G A (SEQ ID NO: 9)Ipomoea nil I T V T G H S L G S (SEQ ID NO: 10)

The senescence-induced lipase protein of the invention was shown topossess lipase activity in both in vitro and in situ assays. For invitro measurements, p-nitrophenylpalmitate and soybean phospholipid (40%phosphatidylcholine and 60% other phospholipids)were used as substrates,and the products of the reactions, p-nitrophenol and free fatty acids,respectively, were measured spectrophotometrically (Pencreac'h andBaratti, 1996; Nixon and Chan, 1979; Lin et al., 1983). Lipase activitywas also measured in vitro by gas chromatography using a modification ofthe method described by Nixon and Chan (1979) and Lin et al. (1983). Thereaction mixture contained 100 mM Tris-HCl (pH 8.0), 2.5 mM substrate(trilinolein, soybean phospholipid or dilinoleylphosphatidylcholine) andenzyme protein (100 μg) in a final volume of 100 μl. The substrates wereemulsified in 5% gum arabic prior to being added to the reactionmixture. To achieve this, the substrates were dissolved in chloroform,added to the gum arabic solution and emulsified by sonication for 30 s.After emulsification, the chloroform was evaporated by a stream of N₂.The reaction was carried out at 25° C. for varying periods of time up to2 hours. The reaction mixture was then lipid-extracted, and the freefatty acids were purified by TLC, derivitized and quantified by GC(McKegney et al., 1995).

Lipolytic acyl hydrolase activity was measure in situ as described byFurukawa et al. (1983) and modified by Tsuboi et al. (1996). In thislatter assay, E. coli transformed with the full length cDNA cloneencoding senescence-induced lipase were grown. in minimal salt mediumsupplemented with Tween 40 or Tween 60, both of which are long chainfatty acid esters, as the only source of carbon. Thus, carbon forbacterial growth was only available if the fatty acid esters werehydrolyzed by lipase. The finding that E. coli transformed with thescenescence-induced lipase cDNA grow in Tween 40- and Tween 60-basalmedium after an initial lag phase, whereas control cultures of E. colithat were not transformed do not grow, confirms the lipase activity ofthe encoded recombinant protein (FIG. 5). That is, thesenescence-induced lipase releases stearate (Tween 60) and palmitate(Tween 40) to obtain the necessary carbon for growth.

“Functional derivatives” of the senescence-induced lipase protein asdescribed herein are fragments, variants, analogs, or chemicalderivatives of senescence-induced lipase, which retain at least aportion of the senescence-induced lipase activity or immunological crossreactivity with an antibody specific for senescence-induced lipase. Afragment of the senescence-induced lipase protein refers to any subsetof the molecule. Variant peptides may be made by direct chemicalsynthesis, for example, using methods well known in the art. An analogof senescence-induced lipase refers to a non-natural proteinsubstantially similar to either the entire protein or a fragmentthereof. Chemical derivatives of senescence-induced lipase containadditional chemical moieties not normally a part of the peptide G38 orpeptide fragment. Modifications may be introduced into thesenescence-induced lipase peptide or fragment thereof by reactingtargeted amino acid residues of the peptide with an organic derivatizingagent that is capable of reacting with selected side chains or terminalresidues.

A senescence-induced lipase protein or peptide according to theinvention may be produced by culturing a cell transformed with anucleotide sequence of this invention (in the sense orientation),allowing the cell to synthesize the protein and then isolating theprotein, either as a free protein or as a fusion protein, depending onthe cloning protocol used, from either the culture medium or from cellextracts. Alternatively, the protein can be produced in a cell-freesystem. Ranu, et al., Meth. Enzymol., 60:459-484, (1979).

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting tothe present invention.

EXAMPLE 1

Plant Materials Used To Isolate The Carnation Lipase cDNA

Carnation plants (Dianthus caryophyllus L. cv. Improved white Sim) grownand maintained in a greenhouse were used to isolate the nucleotidesequence corresponding to the senescence-induced lipase gene. Flowertissue in the form of senescing flower petals (from differentdevelopmental stages) was collected in buffer or stored at −70° C. untilused.

Cytosolic lipid particles were isolated from carnation flower petalsharvested just before the onset of senescence. Carnation petals (25g/150 ml buffer) were homogenized at 4° C. in homogenization buffer (50mM Epps- 0.25 M sorbitol pH 7.4, 10 mm EDTA, 2 mM EGTA, 1 mM PMSF, 1 mMbenzamadine, 10 mm amino-n-caproic acid and 4% polyvinylpolypyrrolidone)for 45 seconds in an Omnimizer and for an additional minute in aPolytron homogenizer. The homogenate was filtered through four layers ofcheesecloth, and the filtrate was centifuged at 10,000 g for twentyminutes at 4° C. The supernatant was centrifuged for one hour at 250,000g to isolate microsomal membranes. The lipid particles were obtainedfrom the post-microsomal supernatant by collecting the particles afterfloatation centrifugation by the method of Hudak and Thompson, (1997),Physiol. Plant., 114:705-713. The supernatant was made 10% (w/v) withsucrose, and 23 ml of the supernatant were poured into 60 Ti Beckmancentrifuge tubes, overlayed with 1.5 ml isolation buffer and centrifugedat 305,000 g for 12 hours at 4° C. The particles were removed from theisolation buffer overlayer with a Pasteur pipette. Three ml of particlesuspension were loaded onto a Sepharose G-25 column equilibrated withsterile PBS (10 mM sodium phosphate buffer pH 7.5 plus 0.85% sodiumchloride) and the suspension was eluted with sterile PBS. The voidvolume containing the particles was eluted and concentrated using aCentricon-10 filter (available from Amicon) to a protein concentrationof 600 μg. The lipid particles were then used to generate antibodies inrabbits inoculated with 300 μg of the particles. The IgG titer of theblood was tested by Western blot analysis.

Messenger RNA (mRNA) Isolation

Total RNA was isolated from petals of stage I, II, III or IV carnationflowers essentially as described by Chomczynski and Sachi, Anal.Biochem., 162:156-159 (1987). Briefly, 15 g of petal tissue were frozenin liquid nitrogen and homogenized for 30 seconds in buffer containing 4M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarkosyland 0.1 M β-mercaptoethanol. 150 ml water-saturated phenol, 30 ml ofchloroform and 15 ml of 2 M NaOAc, pH 4.0 were added to the homogenizedsample. The sample was centrifuged at 10,000 g for ten minutes and theaqueous phase removed and nucleic acids precipitated therefrom with 150ml isopropanol. The sample was centrifuged for ten minutes at 5,000 gand the pellet was washed once with 30 ml of 4 M LiCl, extracted with 30ml chloroform and precipitated with 30 ml isopropanol containing 0.2 MNaOAc, pH 5.0. The RNA was dissolved in

DEPC-treated water and stored at −70° C.

PolyA⁺ mRNA was isolated from total RNA using the PolyA³⁰ tract mRNAIsolation System available from Promega. PolyA⁺ mRNA was used as atemplate for cDNA synthesis using the ZAP Express° cDNA synthesis systemavailable from Stratagene (La Jolla, Calif.)

Carnation Petal cDNA Library Screening

A cDNA library made using mRNA isolated from stage IV carnation petalswas diluted to approximately 5×10⁶ PFU/ml and immunoscreened with lipidparticle antiserum. Positive cDNA clones were recovered using theExAssist® Helper Phage/SOLR strain system and recircularized in apBluescript® phagemid (Stratagene). A stage III carnation petal cDNAlibrary was also screened using a ³²P-labelled 19 base pair probe(5′-ACCTACTAGGTTCCGCGTC-3′) (SEQ ID NO:5). Positive cDNA clones wereexcised from the phages and recircularized into a pBK-CMV® (Stratagene)phagemid using the method in the manufacturer's instructions. The fulllength cDNA (1.53 kb fragment) was inserted into the pBK-CMV vector.

Arabidopsis Leaf cDNA Library Screening

A full-length cDNA clone (1338 bp) of the senescence-induced lipase genefrom Arabidopsis thaliana was isolated by screening an Arabidopsissenescent leaf cDNA library. The probe used for screening the librarywas obtained by PCR using the scenescent leaf library as template. ,ThePCR primers were designed from the genomic sequence (U93215) present inGenBank. The forward primer had the sequence 5′ ATG TCT AGA GAA GAT ATTGCG CGG CGA 3′ (SEQ ID NO:20) and the reverse primer had the sequence 5′GAT GAG CTC GAC GGA GCT GAG AGA GAT G 3′ (SEQ ID NO:21). The PCR productwas subcloned into Bluescript for sequencing. The nucleotide and aminoacid sequence of the PCR product used are shown in FIG. 14.

Plasmid DNA Isolation, DNA Sequencing

The alkaline lysis method described by Sambrook et al., (Supra) was usedto isolate plasmid DNA. The full length positive cDNA clone wassequenced using the dideoxy sequencing method. Sanger, et al., Proc.Natl. Acad. Sci. USA, 74:5463-5467. The open reading frame was compiledand analyzed using BLAST search (GenBank, Bethesda, Md.) and alignmentof the five most homologous proteins with the derived amino acidsequence of the encoded gene was achieved using a BCM Search Launcher:Multiple Sequence Alignments Pattern-Induced Multiple Alignment Method(See F. Carpet, Nuc. Acids Res., 16:10881-10890, (1987)). Functionalmotifs present in the derived amino acid sequence were identified byMultiFinder.

Expression Of The Lipase As A Fusion Protein

Phagemid pBK-CMV containing the full length carnation senescence-inducedlipase was digested with EcoRI and XbaI, which released the 1.53 Kblipase fragment, which was subcloned into an EcoRI and XbaI digestedfusion vector, pMalc (New England BioLabs). , The pMalc vectorcontaining the senescence-induced lipase, designated pMLip, was used totransform E. coli BL-21(DE3) cells.

The fusion protein encoded by pMLip, (fusion of the senescence-inducedlipase and maltose binding protein) was isolated and purified asdescribed in Sambrook, et al. (Supra)and Ausubel, et al., in CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, New York, (1987), 16.4.1-16.4.3. Briefly, E. coli BL-21cells transformed with pMLip were resuspended in 3 ml/g lysate buffer(50 mM Tris, pH 8.0, 100 mM NaCl and 1 mM EDTA) containing 8 μl of 50 mMPMSF and 80 μl of 20 mg/ml lysozyme per gram of cells and incubated fortwenty minutes at room temperature with shaking. Then, 80 μl of 5%deoxycholic acid and 40 units of DNAse I were added and the cells wereshaken at room temperature until the cells completely lysed. The celldebris was pelleted by centrifugation and resuspended in two volumes oflysate buffer plus 8 M urea and 0.1 mM PMSF. After one hour, sevenvolumes of buffer (50 mM KH₂PO₄, 1 mM EDTA and 50 mM NaCl, pH 7.0) wereadded to neutralize the suspension. The pH of the cell suspension wasadjusted to pH 8.0 with HCl and the cell debris was pelleted. Thesupernatant was dialyzed against 20 mM Tris buffer, pH 8.0, 100 mM NaCland 1 mM EDTA at 4° C. overnight. The maltose binding protein-lipasefusion product (Malip) was purified using an amylose column (availablefrom New England BioLab). Fractions containing the fusion protein werecleaved with Protease Factor Xa (1 μg/100 μg fusion protein) to separatelipase from the fusion product. Both the fusion protein and the cleavedlipase were analyzed by SDS PAGE electrophoresis and Western blots.Maltose binding protein encoded by pMalc was used as a control. Theresults are shown in FIG. 3.

Northern Blot Hybridizations of Carnation RNA

Ten μg of total RNA isolated from flowers at stages I, II, III, IV wereseparated on 1% denatured formaldehyde agarose gels and immobilized onnylon membranes. The 1.53 Kb

EcoRI-XbaI lipase fragment labelled with ³²P-dCTP using a random primerkit (Boehringer Mannheim) was used to probe the filters (7×10⁷ cpm). Thefilters were washed once with 1×SSC, 0.1% SDS at room temperature andthree times with 0.2×SSC, 0.1% SDS at 65° C. The filters were dried andexposed to X-ray film overnight at −70° C. The results are shown in FIG.4.

Northern Blot Hybridization Of Arabidopsis RNA

Ten μg of total RNA isolated from Arabidopsis leaves at weeks 2, 3, 4, 5and 6 of growth were separated on 1% denatured formaldehyde agarose gelsand immobilized on nylon membranes. The full-length Arabidopsissenescence-induced lipase gene labelled with ³²P-dCTP using a randomprimer kit (Boehringer Mannheim) was used to probe the filters (7×10⁷cpm). The filters were washed once with 1×SSC, 0.1% SDS at roomtemperature and three times with 0.2×SSC, 0.1% SDS at 65° C. The filterswere dried and exposed to X-ray film overnight at −70° C.

Genomic DNA Isolation And Southern Blot Hybridizations

Freshly cut carnation petals were frozen in liquid nitrogen, ground to apowder and homogenized (2 ml/g) with extraction buffer (0.1 M Tris, pH8.2, 50 mM EDTA, 0.1M NaCl, 2% SDS, and 0.1 mg/ml proteinase K) toisolate genomic DNA. The homogenized material was incubated at 37° C.for ten minutes and extracted with phenol-chloroform-isoamyl alcohol(25:24:1). DNA was precipitated with NaOAc and isopropanol. The DNApellet was dissolved in 1 x TE, pH 8.0, re-extracted with phenol,reprecipitated and resuspended in 1×TE, pH 8.0.

Genomic DNA was digested with restriction endonucleases (Bam HI, XbaI,XhoI, EcoRI, HindIII and SalI) separately and the digested DNA wasfractionated on a 1% agarose gel. The separated DNA was blotted ontonylon membranes and hybridizations were carried out using³²P-dCTP-labelled 1.53 Kb lipase fragment. Hybridization and washingwere carried out under high stringency conditions (68° C.)). 6×SSC,2×Denhardt's reagent, 0.1% SDS)as well as low stringency conditions (42°C. for hybridization and washing) (6×SSC, 5×Denhardt's reagent, 0.1%SDS). The results are shown in FIG. 6. As can be seen, the lipase cDNAprobe detects only one genomic fragment, indicating that the carnationlipase gene is a single copy gene.

Lipase Enzyme Assays

Lipolytic acyl hydrolase activity of the purified lipase fusion proteinwas assayed spectrophotometrically using p-nitrophenylpalmitate andsoybean phospholipid as exogenous substrates. For maltose-bindingprotein alone, which served as a control, there was no detectable lipaseactivity with phospholipid as a substrate (Table 2). Whenp-nitrophenylpalmitate was used as a substrate with maltose-bindingprotein alone, a small amount of p-nitrophenol, the expected product ofa lipase reaction, was detectable reflecting background levels ofp-nitrophenol in the commercial preparation of p-nitrophenylpalmitate(Table 2). However, in the presence of purified lipase fusion protein,strong lipase activity manifested as the release of free fatty acidsfrom phospholipid and p-nitrophenol from nitrophenylpalmitate wasevident (Table 2).

TABLE 2 Spectrophotometric measurements of the lipolytic acyl hydrolaseactivity of maltose-binding protein and lipase fusion protein expressedin E. coli and purified by amylose column chromatography. PRODUCT pNPPp-nitrophenol free fatty acid Protein Species (nmol/mg/min) (nmol/mgprotein/min) Maltose-binding protein  0.71 ± 0.02 ND* Linase fusionprotein 12.01 ± 1.81 46.75 ± 1.24 Two substrates, p-nitrophenylpalmitateand soybean phospholipid, were used. Activities are expressed in termsof product formed (p-nitrophenol from p-nitrophenylpalmitate and freefatty acid from soybean phospholipid). Means ± SE for n = 3 replicationsare shown. *ND, not detectable

In other experiments, the enzymatic activity of the lipase fusionprotein was assayed by gas chromatography, a technique that enablesquantitation and identification of free fatty acids released from thesubstrate. Trilinolein, soybean phospholipid anddilinoleylphosphatidylcholine were used as substrates, and thedeesterified fatty acids were purified by thin layer chromatographyprior to being analyzed by gas chromatography. In keeping with thespectrophotometric assay (Table 2), there was no detectable lipaseactivity for maltose-binding protein alone with either soybeanphospholipid or dilinoleylphosphatidylcholine, indicating that thesesubstrates are essentially free of deesterified fatty acids (Table 3).However, when the lipase fusion protein was used as a source of enzyme,palmitic, stearic and linoleic acids were deesterified from the soybeanphospholipid extract, and linoleic acid was deesterified fromdilinoleylphosphatidylcholine (Table 3). In contrast to the phospholipidsubstrates, detectable levels of free linoleic acid were present intrilinolein, but the levels of free linoleic acid were significantlyincreased in the presence of lipase fusion protein indicating that thelipase is capable of deesterifying fatty acids from triacylglycerol aswell (Table 3)

TABLE 3 GC measurements of the lipolytic acyl hydrolase activity ofmaltose-binding protein and lipase fusion protein expressed in E. coliand purified by amylose column chromatography Products (μg/mg protein)¹Maltose- Lipase binding fusion Substrates Protein Protein Tri-linolein²Linoleic acid (18:2) 15.9 ± 0.75 33.4 ± 1.58 Soybean Palmitic acid(16:0)  ND⁴ 4.80 phospholipids³ Stearic acid (18:0) ND 9.68 Linoleicacid (18:2) ND 5.80 Dilinoleylphos- Linoleic acid (18:2) ND 20.0phatydilcholine³ ¹Reaction was allowed to proceed for 2 hours, and wasnot continuously linear over this period. ²Means ± SE for n = 3replications are shown ³Single experiment ⁴Not detectable

Lipase activity of the protein obtained by expression of the lipase cDNAin E. coli was measured in vivo as described in Tsuboi, et al., Infect.Immunol., 64:2936-2940 (1996); Wang, et al., Biotech., 9:741-746 (1995);and G. Sierra, J. Microbiol. and Serol., 23:15-22 (1957). A singlecolony of E. coli BL-21 cells transformed.with pMal and another E. coliBL-21 colony transformed with pMLip were inoculated in basal salt medium(pH 7.0) containing (g/L): K₂HPO₄ (4.3), KH₂PO₄ (3.4), (NH₄) SO₄(2.0),MgCl₂(0.16), MnCl_(2 .)4H₂O (0.001), FeSO₄ . 7H₂O(0.0006), CaCl₂ .2H₂O(0.026), and NaMoO₄. 2H₂O (0.002). Substrate, Tween 40(polyoxyethylenesorbitan monopalmitate) or Tween 60(polyoxyethylenesorbitan monostearate), was added at a concentration of1%. Growth of the bacterial cells at 37° C. with shaking was monitoredby measuring the absorbance at 600 nm (FIG. 5). As can be seen in FIG.5, E. coli cells transformed with pMLip were capable of growth in theTween40/Tween60-supplemented basal medium, after an initial lag period.However, E. coli cells transformed with pMal did not grow in theTween-supplemented medium.

EXAMPLE 2

Ethylene Induction of Carnation Senescence-Induced Lipase Gene

Stage II carnation flowers and carnation cuttings were treated with 0.5ppm ethylene in a sealed chamber for 15 hours. RNA was extracted fromthe ethylene treated Stage II flower petals and from leaves of thetreated cutting, as well as from the flower and leaves of untreatedcarnation flowers and cuttings as described below.

Arabidopsis plants were treated with 50 μM ethephon in a sealed chamberfor one, two or three days. RNA was extracted from the ethephon treatedleaves of the plants as follows.

Flowers or leaves (1 flower or 5 g leaves) were ground in liquidnitrogen. The ground powder was mixed with 30 ml guanidinium buffer (4 Mguanidinium isothiocyanate, 2.5 mM NaOAc pH 8.5, 0.8%β-mercaptoethanol). The mixture was filtered through four layers ofcheesecloth and centrifuged at 10,000 g at 4° C. for 30 minutes. Thesupernatant was then subjected to cesium chloride density gradientcentrifugation at 26,000 g for 20 hours. The pelleted RNA was rinsedwith 75% ethanol, resuspended in 600 μl DEPC-treated water and the RNAprecipitated at −70° C. with 0.75 ml 95% ethanol and 30 μl of 3M NaOAc.Ten μg of either carnation or Arabidopsis RNA were fractionated on a1.2% denaturing formaldehyde agarose gel and transferred to a nylonmembrane. Randomly primed ³²P-dCTP-labelled full length carnation lipasecDNA (SEQ ID NO:1) was used to probe the membrane containing carnationRNA at 42° C. overnight. Randomly primed ³²P-dCTP-labelled full lengthArabidopsis lipase cDNA was used to probe the membrane containingArabidopsis RNA at 42° C. overnight. The membranes were then washed oncein 1×SSC containing 0.1% SDS at room temperature for 15 minutes andthree times in 0.2×SSC containing 0.1% SDS at 65° C. for 15 minuteseach. The membranes were exposed to x-ray film overnight at −70° C.

The results are shown in FIG. 9 (carnation) and FIG. 16 (Arabidopsis;lane 1, one day treatment; lane 2, two days treatment; lane 3, threedays treatment). As can be seen, transcription of the carnation lipaseand Arabidopsis lipase is induced in flowers and/or leaves by ethylene.

EXAMPLE 3

Generation of Tomato PCR Product Using Carnation Lipase Primers

A partial length senescence-induced lipase sequence from tomato genomicDNA obtained from tomato leaves was generated by nested PCR using a pairof oligonucleotide primers designed from carnation senescence-inducedlipase sequence. The 5′ primer is a 19-mer having the sequence,5′-CTCTAGACTATGAGTGGGT (SEQ ID NO:7); the 3′ primer is an 18-mer havingthe sequence, CGACTGGCACAACCTCCA-3′ (SEQ ID NO:8). Polymerase chainreaction, using genomic tomato DNA was carried out as follows:

Reaction components: Genomic DNA 100 ng dNTP (10 mM each) 1 μl MgCl₂ (5mM) + 10x buffer 5 μl Primers 1 and 2 (20 μM each) 0.5 μl Taq DNApolymerase 1.25 U Reaction volume 50 μl Reaction parameters: 94° C. for3 min 94° C./1 min, 48° C./1 min, 72° C./2 min, for 45 cycles 72° C. for15 min.

The tomato partial length sequence obtained by PCR has the nucleotidesequence, SEQ ID NO:6 (FIG. 10) and a deduced amino acid sequence as setforth in FIG. 10. The partial length sequence contains an intron (FIG.10, lower case letters) interspersed between two coding sequences. Thetomato sequence contains the conserved lipase consensus sequence,ITFTGHSLGA (SEQ ID NO:3).

The tomato sequence has 53.4% sequence identity with the carnationsenescence-induced lipase sequence and 43.5% sequence identity withArabidopsis lipase, the latter of which has 44.3% sequence identity withthe carnation sequence.

EXAMPLE 4

Effect Of Chilling On Cell Membrane Integrity In Tomato Plants

Tomato plants were chilled for 48 hours at 7° C. to 8° C. and thenreturned to room temperature for 24 hours. The effect of chilling onleaves was assessed by measuring the amount of electrolyte leakage(μMhos).

Specifically, 1 g of leaf tissue was cut into a 50 ml tube, quick-rinsedwith distilled water, and 40 ml of deionized water added. The tubes werecapped and rotated at room temperature for 24 hours. Conductivity (μMho)readings reflecting electrolyte leakage were taken at 6 and 24 hourintervals for control and chill-injured leaf tissue. It is clear fromFIG. 11 that electrolyte leakage reflecting membrane damage is incurredduring the rewarming period in chill injured leaf tissue.

Northern Blot Analysis Of RNA Obtained From Chilled Tomato Leaves

Total RNA was isolated from the leaves 15 g of unchilled tomato plants(control) and chilled tomato plants that had been returned to roomtemperature for 0, 6 and 24 hours. RNA extraction was carriedout asdescribed in Example 3. 10 μg of RNA from each sample was separated on a1.2% denaturing formaldehyde gel and transferred to a nylon membrane.The membrane was probed with ³²P-dCTP-labelled probe (SEQ ID NO:3)andthen washed under the same conditions as described in Example 3. Theresults are shown in FIG. 12.

As can be seen from the autoradiograph (FIG. 12B) tomato lipase geneexpression is induced by chilling and the pattern of gene inductioncorrelates with increased electrolyte leakage in chill injured leaves(FIG. 11).

EXAMPLE 5

Generation Of Transgenic Plants Expressing Senescence-Induced LipaseGene In Antisense Orientation

Agrobacteria were transformed with the binary vector, pKYLX71,containing the full-length Arabidopsis senescence-induced lipase geneexpressed in antisense orientation under the regulation of double 35Spromoter. Arabidopsis plants were transformed with these Agrobacteria byvacuum filtration, and transformed seeds from the resultant T₀ plantswere selected on ampicillin.

T₁ plants were grown under greenhouse conditions, alongside wild-typeArabidopsis plants. Differences in leaf size, overall plant size, seedyield and leaf senescence between transgenic and wild-type plants wereobserved over time. Differences are illustrated in FIGS. 17, 18, 19, and20.

EXAMPLE 6

Reduced Senescence-Induced Lipase Production In Transgenic Plants

Total protein isolated from leaves of four week-old Arabidopsiswild-type and corresponding transgenic plants made as in example 5 wastransferred to a nylon membrane and probed with antibody raised againstthe Arabidopsis senescence-induced lipase protein. The Western blot isshown in FIG. 21.(Lanes 1 and 2 were loaded with 9 μg of protein, andlanes 3 and 4 were loaded with 18 μg of protein). The expression of thesenescence-induced lipase was reduced in transgenic plants.

1: An isolated DNA molecule encoding senescence-induced lipase, whereinthe DNA molecule hybridizes under low stringency conditions with SEQ IDNO: 1, SEQ ID NO: 18 or both, or a functional derivative of the isolatedDNA molecule which hybridizes with SEQ ID NO: 1, SEQ ID NO: 18 or both.2-53. (canceled)